Discovery of a non-canonical prototype long-chain monoacylglycerol lipase through a structure-based endogenous reaction intermediate complex

The identification and characterization of enzyme function lags behind the rapidly increasing availability of protein sequences and structures. This is often due to a lack of knowledge about in vivo relevant substrates. In vitro studies and purified protein samples often fail to capture the full substrate profile of an enzyme within its natural cellular environment. While high-resolution structural biology offers a powerful tool for visualizing enzyme-ligand complexes, it requires pure compounds, which are not always available, especially for large hydrophobic substrates. This study demonstrates the power of using endogenous substrate uptake for the structural and functional characterization of an orphan enzyme with putative lipase/esterase activity, focusing on an enzyme from the thermophilic anaerobic bacterium *Thermoanaerobacter thermohydrosulfuricus* (Tth). This enzyme shows remarkable robustness against various solvents and temperatures, making it an attractive target for biotechnology applications, but its molecular mechanisms remained unclear. Lipases are crucial enzymes in various industrial processes due to their ability to catalyze the hydrolysis and synthesis of glycerol esters. Their activity mainly occurs at water-lipid interfaces, a process known as interfacial activation, which is often facilitated by an additional α-helical domain called the "lid" or "cap". However, the lid's diversity and lack of sequence conservation make it challenging to predict substrate specificity. The study aimed to overcome the limitations of traditional methods by leveraging the enzyme's complex with an endogenous reaction intermediate obtained directly from its expression host, providing a unique perspective on its function and substrate specificity.

Extensive research has been conducted on lipases and their applications in biotechnology. Various studies have explored lipase-catalyzed processes in diverse fields such as medical biotechnology, detergent industry, organic synthesis, biodiesel production, agrochemical industry, flavor and aroma industry, and food production. However, the understanding of lipase function in its native environment has been limited due to difficulties in working with insoluble long-chain glycerol esters under standard experimental conditions. Previous molecular activity studies have primarily focused on isolated or recombinant lipases, often lacking the complexity of the cellular context. The thermophilic nature of the Tth enzyme and its extreme environment of origin suggested a unique adaptation and potential for novel catalytic properties. Existing computational methods for enzyme function prediction, while powerful, have limitations in proving function in vivo. This study adds to the body of knowledge by using a different approach, focusing on an in vivo interaction to determine function.

The study employed a multi-faceted approach combining structural biology, biochemical assays, and genetic manipulation. The Tth MAG lipase was expressed and purified from *E. coli*, and its oligomeric state was analyzed using size exclusion chromatography (SEC). Thermal stability was assessed by nano differential scanning fluorimetry (nanoDSF). High-resolution crystal structures were determined using X-ray crystallography, both in the presence and absence of the inhibitor phenylmethylsulfonyl fluoride (PMSF). The crystals were analyzed by reversed-phase liquid chromatography-mass spectrometry (LC-MS) to identify the bound ligand. Site-directed mutagenesis was used to generate various enzyme variants to investigate the roles of specific residues in catalysis. The enzymatic activity of the wild-type enzyme and its variants was quantitatively assessed using in vitro assays with a range of substrates, including monoacylglycerols (MAGs), diacylglycerols (DAGs), triacylglycerols (TAGs), and *p*-nitrophenyl (pNP) esters of varying acyl chain lengths. Michaelis-Menten kinetics was used to determine kinetic parameters such as K_M, k_cat, and k_cat/K_M. Finally, the release of fatty acids (FAs) by different Tth MAG lipase variants expressed in *E. coli* was analyzed using gas chromatography-mass spectrometry (GC-MS) and high-performance thin-layer chromatography (HPTLC) to assess the in vivo activity. Reductive methylation was performed on Tth MAG lipase to improve crystal quality, and the effect of the methylation on activity was assessed.

The high-resolution crystal structures of Tth MAG lipase, both active and PMSF-inhibited, revealed a two-fold symmetric dimer with a minimal lid domain comprising an α-helix/β-hairpin/α-helix (HBH) topology. An endogenous C18 MAG reaction intermediate was identified bound in the active site, its distal part residing in a hydrophobic tunnel within the lid domain. In vitro assays demonstrated high specificity for MAG substrates over DAG and TAG esters, with significant activity observed for C8 MAG. Mutagenesis studies highlighted the crucial role of the active site Ser113, the lid domain, and Glu43 in catalysis. The Glu43 residue was found to directly interact with the glycerol moiety, contributing to the high MAG specificity. Modifications to Tyr154 within the lid tunnel had only minor effects on activity. Steady-state Michaelis-Menten kinetics using pNP esters revealed an increase in binding affinities with increasing acyl chain length and consistent catalytic efficiencies for medium-chain length substrates. Analysis of FA composition in *E. coli* expressing different Tth MAG lipase variants showed a correlation between the presence of the enzyme and FA release, further supporting its in vivo activity as a MAG lipase. Sequence analysis revealed that Tth MAG lipase belongs to a family of HBH lid-containing lipases/esterases with distinct substrate specificities, primarily from bacterial and archaeal sources. A structural comparison with related enzymes, such as cinnamoyl esterase from *Lactobacillus johnsonii* and feruloyl esterase from *Butyrivibrio proteoclasticus*, revealed an explanation for the different substrate specificities due to the structure of the lid.

This study successfully elucidated the function of an orphan enzyme using a structure-based approach that utilized the endogenous reaction intermediate. The findings demonstrate that the minimal HBH lid domain in Tth MAG lipase is sufficient for the turnover of long-chain MAG esters, providing a prototype for a growing family of lipases. The combination of in vitro and in vivo data provides a comprehensive understanding of the enzyme's catalytic mechanism and substrate specificity. The results suggest that a deep understanding of MAG lipase catalysis requires a detailed understanding of both the active site and the lid domain's structure and dynamics. The findings expand the knowledge of HBH lid lipases and highlight their potential for evolving to recognize a wider range of lipidic substrates. The success in capturing the endogenous complex highlights the importance of considering the native cellular environment for understanding enzyme function.

This study successfully characterized a novel long-chain MAG lipase using high-resolution structural biology and biochemical assays. The findings reveal a minimal prototype of HBH lid-containing lipases with a unique catalytic mechanism involving a hydrophobic tunnel within the lid domain for substrate binding. This work demonstrates the power of capturing endogenous enzyme-substrate complexes for functional annotation and expands our understanding of lipase catalysis and its potential biotechnological applications. Future research could focus on exploring the full range of substrates and designing improved variants for industrial applications.

The study focused primarily on the characterization of Tth MAG lipase under specific conditions. The generalizability of the findings to other MAG lipases requires further investigation. The use of pNP esters as substrates for kinetic analysis might not perfectly reflect the activity with natural MAG substrates. Further analysis is needed to fully understand the role of the dimeric interface in catalysis. The in vivo experiments were conducted in E.coli, which may not fully replicate the natural environment of the enzyme.